The overall goal is to elucidate the mechanism by which prostanoid synthesis in blood and vascular cells is regulated under pathophysiological stimuli and to develop pharmacological and genetic strategies to control excessive prostanoid synthesis. During the current funding period, we have made novel observations regarding the transcriptional control of cyclooxygenase-2 (COX-2) by endogenous mechanism and exogenous compounds. We observed that proliferating fibroblasts release a factor that suppressed COX-2 expression in human endothelial cells, fibroblasts and murine macrophages stimulated with diverse agonists. This factor which is named cytoguardin has been purified to near homogeneity. Preliminary results indicate that cytoguardin inhibits COX-2 transcription by blocking p300 coactivator interaction with COX-2 promoter-bound transactivators. In this renewal application we propose to purify cytoguardin to homogeneity by multiple chemical and biochemical procedures, identify its chemical structure by NMR spectrometry, investigate its activity and elucidate the mechanism of its action. We will carry out gene expression profiling by microarray to determine the cluster of cellular genes that are suppressed by this endogenous transcriptional regulator. The new findings will revolutionize our view about gene control and cell defense and will be valuable for new drug development. Another important observation is that aspirin and sodium salicylate suppressed COX-2 transcription by inhibiting C/EBPI3 binding to its cognate site on the proximal COX-2 promoter region. Preliminary results suggest that salicylates act on a kinase that phosphorylate C/EBP]3, thereby inhibiting its binding to promoter. We propose to use combinatorial approaches to identify the kinase and elucidate the mechanism by which salicylates suppress the kinase activity. Findings from this subproject will solve a pharmacological dilemma and provide new insight into designing new anti-inflammatory and anti-neoplastic drugs. Since COX-2 plays a critical role in pathologic angiogenesis, we postulated that cytoguardin and salicylate should be able to suppress angiogenesis via blocking COX-2 expression. Our preliminary data show that aspirin and sodium salicylate inhibited endothelial tube formation induced by VEGF and cancer cells in a concentration-dependent manner. Semi-purified cytoguardin abrogated endothelial tube formation induced by VEGF and cancer cells. We propose to confirm these in vitro experiments by evaluating the effects of cytoguardin and salicylates on angiogenesis in animal models. Furthermore, the mechanisms by which cytoguardin and salicylate inhibit COX-2 mediated angiogenesis will be elucidated. Information provided by studies from this renewal application will have great impact on understanding the fundamental cytoprotection process and offer an excellent opportunity for developing new therapeutic agents.